Tracheal epithelial cells play a vital role in pulmonary host defense by clearing foreign materials from the airways through the process of mucociliary clearance. Despite this critical role, our understanding of the cellular and molecular mechanisms responsible for the regulation of mucociliary clearance is far from complete. The broad, long-term hypothesis of this project is that effective clearance depends on cooperative actions and coordinated responses of ciliated cells and mucus-secreting cells. We refer to this as 'secretion-clearance coupling'. Parasympathetic cholinergic input to the airway, acting via muscarinic receptors, plays an important role in regulating mucociliary clearance and these receptors are found on both ciliated cells and submucosal glands. In this proposal, we seek to understand the mechanisms responsible for the regulation of ciliary beating frequency (CBF) by acetylcholine (ACh). We hypothesize that ACh, acting through m3 muscarinic receptors on ciliated cells, has dual, opposing actions on CBF and, further, that these actions are mediated by changes in cytoplasmic free Ca2+ concentration [Ca2+]i resulting from ACh activation of this G-protein coupled receptor. The net result of these opposing actions serves to give the ciliated cell an extraordinary degree of fine control in regulating CBF responses. In order to test this hypothesis, we will examine, using high resolution digital video microscopy, mechanisms by which ACh can raise and lower [Ca2+]i, measure the tightness of coupling between Ca2+ and CBF, and begin to identify signaling molecules that mediate such coupling. The specific aims are: I) To define the mechanisms responsible for regulating [Ca2+]i in ciliated cells in response to ACh. II) To characterize the kinetics of coupling between [Ca2+]i and CBF. Ill) To characterize the molecular site of Ca2+ action. An increased understanding of the normal mechanisms that regulate ciliary beating will help to define the basis for defective mucociliary clearance in such common diseases as asthma and chronic bronchitis. Present day therapy for mucociliary dysfunction is unsatisfactory. The proposed experiments will help to identify molecular targets that might serve as the basis for novel treatment strategies for such airway diseases.